The present invention relates to a process for the isolation of p-benzosemiquinone of formula 1: 
a major harmful oxidant from cigarette smoke. More particularly the present invention provides a process for the isolation of p-benzosemiquinone, a major harmful oxidant from cigarette smoke, which is responsible for the oxidative damage of proteins and DNA
Exposure to cigarette smoke is a major cause of life-threatening diseases like bronchitis, emphysema, other diseases of the respiratory tract, coronary heart diseases, lung cancer and other malignancies [1-5]. In fact, cigarette smoke is the overwhelming cause of lung cancer, now the most common cancer globally. Since approaches to cessation of smoking by public health campaigns and anti-smoking laws passed by local Governments have had limited success, the most practicable approach is the prevention of the hazardous effects caused by cigarette smoke. Cigarette smoke in known to contain about 4000 components, out of which about 3000 components are present in the gas phase and about 1000 components in the tar phase [6]. The oxidants in the gas phase, such as O2xe2x88x92, H2O2, NO, peroxy radical are extremely unstable [7]. If the gas phase is passed into phosphate buffer and the resultant solution is added to albumin solution, no protein oxidation occurs (7). Apparently, any damage caused by the gas phase is expected to be restricted to the buccal cavity and upper respiratory tract [8]. On the other hand, the oxidant(s) present in the tar are quite stable and these are apparently responsible for producing oxidative damage in the lung, heart and other organs [7,9]. About 48 percent of the tar components are water soluble [10] and the aqueous extract of tar is known to produce oxidative damage of biological macromolecules including proteins and DNA [7,11,12]. However, it is perplexing to conceive how many of the components present in the aqueous extract of tar are responsible for producing oxidative damage in the biological system. Uptil now, among the many components of cigarette smoke, three classes of compounds have been suggested to be implicated as causative agents in the development of cancer and degenerative diseases, namely, (i) polycyclic aromatic hydrocarbons (ii) nitrosamines and (iii) free radicals.
Among the polycyclic hydrocarbons, benzo [a] pyrene is by far the best studied. But it is not a carcinogen and requires metabolic activation through cytochrome P450 system to become the ultimate carcinogen, benzo [a] pyrene diol epoxide. Moreover, the concentration of benzo [a] pyrene in cigarette smoke is meagre, about 10 to 40 ng per cigarette [13] and benzo [a] pyrene cannot explain oxidative damage of protein produced by cigarette smoke.
Among the tobacco specific nitrosamines (TSNA), the most studied ones are N1-nitrosonornicotine (NNN) and 4-(methylnitrasamino)-1-(3-pyridyl)-1-butanone (NNK). Again TSNA are not direct carcinogens and also their concentrations in tobacco smoke vary widely. The observed range for NNN is 0.004 xcexcg to 1.35 xcexcg and for NNK,  less than 0.004 xcexcg to 1.75 xcexcg per cigarette. It is concluded that TSNA in cigarette smoke is not a sufficient index for the carcinogenic potential of cigarette smoke [14]. Again TSNA cannot explain oxidative damage of proteins.
Another aspect of the hazardous component of cigarette smoke is free radical. Pryor and his associates made considerable studies on free radical chemistry of cigarette smoke and its toxicological implications. These authors suggest that the principal relatively stable free radical in cigarette tar may be a quinone/hydroquinone complex which is an active redox system and that this redox system is capable of reducing molecular oxygen to produce superoxide, leading to hydrogen peroxide and hydroxyl radicals [15], that may eventually lead to oxidative damage of biological macromolecules but we have observed that oxidative damage of proteins produced by the stable tar radicals is not inhibited by SOD or catalyst indicating that the oxidative damage is not mediated by super oxide radical or hydrogen peroxide. The applicants have further observed that the tar radicals oxidize proteins in nitrogen atmosphere and in the absence of molecular oxygen, indicating a direct interaction of the tar radicals with biological micromolecules. However, these authors admit that the principal radical they have identified in tar is actually not a monoradical and probably is not a single species (16). They also admit that cigarette tar is an incredibly complex mixture and since the tar radicals have not been isolated and unambiguously identified, any conclusion concerning the chemistry or biochemistry of the tar radicals must be regarded as tentative [15].
It is noteworthy to mention that by the 1960s, the tobacco industry in general had proven in its own laboratory that cigarette tar causes cancer in animals [17]. Throughout 1960s the companies"" researchers tried to discover the toxic elements in cigarette smoke with the conviction that if the toxic components could be identified, these agents could be removed or eliminated and a xe2x80x9csafexe2x80x9d cigarette could be created, which would deliver nicotine without delivering the toxic substances [17]. But by the late 1970s, the tobacco industry had largely abandoned this particular research, because the objective proved to be unattainable. It was a problem technically difficult to solve and proved untractable [17].
Very recently, we have observed that aqueous extract of whole cigarette smoke/tar contains a major harmful oxidant in relatively high amount, approximately 190xc2x110 xcexcg per cigarette. The applicants have isolated the oxidant, determined the structure and found it to be p-benzosemiquinone. The oxidant almost quantitatively accounts for the oxidative damage of proteins produced by the aqueous extract of whole cigarette smoke/tar. The oxidant is also responsible for DNA oxidation. Nagata et al. (18) have shown that semiquinone radicals bind to DNA and damage it. It is also known that oxidative damage of DNA is implicated with mutation and cancer. The oxidant is relatively stable. Its half-life in the solid state at room temperature is approximately 48 hours. The presence of the stable oxidant in cigarette smoke would explain the deleterious effects of side stream smoke and passive smoking (7). The oxidant is absent in nonsmoking tobacco and is produced during burning of the cigarette (7). Applicants have identified a number of chemical compounds/agents those deactivate the oxidant and may be used as antidotes.
Main object of the present invention relates to isolation and characterization of a major harmful oxidant from aqueous extract of whole cigarette smoke/tar, which is mainly responsible for the oxidative damage of biological macromolecules including proteins and DNA.
Another object of the invention is to provide a method for the quantitative assay of cigarette smoke (cs) oxidant present in the whole cigarette solution
Still another object of the invention is to the identification of chemical compounds/agents those will deactivate the oxidant and act as antidotes for combating the harmful effect of the oxidant.
A relatively stable major harmful oxidant has been isolated from aqueous extract of whole cigarette smoke/tar and purified to the extent of  greater than 99% by differential solvent extraction, thin layer chromatography and preparative HPLC. The yield is about 16 xcexcg per cigarette, which is about 8.4% of the amount (≈190 xcexcg) present in the smoke of one cigarette. Comparable results were obtained from twelve different brands of commercial cigarettes. The purified oxidant crystallizes in fine needle shaped very pale yellow crystals from a solution in acetone. The structure of the oxidant has been found to be p-benzosemiquinone as evidenced by elemental analysis, mass spectrum, UV, fluorescence, IR, H-NMR, C-NMR and ESR spectroscopy as well as by chemical properties. The oxidant can be measured quantitatively by either UV absorption spectroscopy or HPLC.
In p-benzosemiquinone, the unpaired electron is delocalised over an aromatic framework containing heteroatoms leading to different mesomeric forms, namely, anionic, neutral and cationic forms (FIG. 1, see ref. 19). This resonance would explain the stability of the semiquinone. The half-life of the oxidant stored in the solid state at the room temperature in air and under darkness is about 48 hours as determined by its capacity to oxidize ascorbic acid. In aqueous solution at pH 7.4, the half-life is about 1.5 hours. Using oxidation of BSA or oxidative degradation of guinea pig lung microsomal proteins as model systems, the oxidant quantitatively accounts for the oxidative damage produced by the aqueous extract of whole cigarette smoke. The cs-oxidant is also responsible for DNA oxidation.
A number of chemical compounds/agents have been identified those inactivate the oxidant and act as antidotes.
Accordingly, the present invention provides a process for the isolation of p-benzosemiquinone of formula 1, a harmful oxidant and the compound identified to counteract the harmful effect caused by this oxidant. 
In an embodiment of the present invention, provides a process for isolating the major harmful oxidant from cigarette smoke responsible for the oxidative damage of proteins and DNA, the said process comprising the steps of:
a) obtaining tar solution from lighted conventional filter tipped cigarette in a glass flask dipped in a mixture of ice and salt;
b) allowing the tar to condense and settle at the bottom of the flask to obtain whole cs solution;
c) extracting the said tar with 30-60 mM potassium phosphate buffer at a pH ranging between 7.4 to 7.8;
d) filtering the solution of step (c) through 0.45 xcexcm Millipore filter;
e) adjusting the pH of the filtrate obtained from step (d) by aqueous NaOH solution to obtain the desired cigarette smoke aqueous extract solution,
f) extracting the above said cs aqueous solution thrice using equal volume of methylene chloride, discarding the lower methylene chloride layer and collecting the upper yellow colored semi purified extract of cigarette smoke solution;
g) further extracting the aqueous extract of cigarette smoke of step (f) twice using equal volume of water-saturated n-butanol, pooling yellow n-butanol extract and lyophilizing at a temperature ranging between xe2x88x9250xc2x0 C. to xe2x88x9260xc2x0 C. under vacuum;
h) extracting the lyophilized material of step (g) twice using HPLC grade acetone to obtain acetone soluble extract;
i) drying the acetone soluble extract of step (h) under vacuum to yield a residue;
j) dissolving the residue of step (i) in HPLC grade methanol;
k) subjecting methanol solution of steps) to preparative TLC using non-fluorescent silica plates, developing the said silica plates using a solvent system constituting mixture of toluene and ethyl acetate in a ratio of 80:20, taking out the plate, drying at about 25-30xc2x0 C. using a drier, cutting small strips containing the developed material from both sides of the plates and keeping them in an iodine chamber for the location of the band corresponding to Rf 0.26, scraping the band and extracting the band material with HPLC grade acetone, filtering and collecting the acetone solution and drying under vacuum to get a pale yellow residue; and
l) dissolving the residue of step (k) by adding equal volume of milli Q water, extracting the aqueous solution with equal volume of HPLC grade water saturated n-butanol and finally followed by drying upper n-butanol layer in small glass tubes under vacuum to obtain the major cigarette smoke (cs) oxidant with a purity of 98-99% and yield of 18-22 xcexcg per cigarette,
In an embodiment of the present invention, wherein said cs oxidant obtained from step (l) is further purified by HPLC after dissolving in a mobile phase comprising a mixture of methylene chloride and methanol in a ratio of 90:10 (v/v) and injecting it in a HPLC instrument with a normal phase 25 cm silica column using a UV detector at 294 nm at a flow rate of 0.5 ml/min, at a temperature of about 25xc2x0 C., at a pressure of about 29 kgf/cm2, collecting the effluent which appears as a single peak at a retention time of 8.808 min with a purity of 100% and yield of about 8.4% of the total cs oxidant (p-benzosemiquinone) present in the parent cs solution.
In another embodiment of the invention, wherein primary cs solution of step (a) is also obtained from lighted convention filter cigarette by passing the whole cigarette smoke into 30-60 mM potassium buffer at pH 7.4-7.8, filtering the above solution through 0.45 xcexcm Millipore filter, adjusting the pH to 7.4 to 7.6 of the filtrate by adding aqueous NaOH solution and performing steps (b) to (l) for obtaining the major cs oxidant p-benzosemiquinone.
Another embodiment of the invention, wherein the said isolated pure cigarette smoke (cs) oxidant p-benzosemiquinone has the following characteristics:
a) on crystallizing with acetone to form small faint yellow needle crystals, having pungent smell similar to that of rancid butterfat,
b) UV absorption maxima in methanol solution are at 293.4 nm and 223.0 nm and in aqueous solution are in 288 nm and 221 nm respectively,
c) on excitation at 293 nm in methanol solution the observed emission maxima are at 329.6 nm and 651.4 nm and on excitation at 224 nm, the observed emission maxima are at 329.6 nm and 652.6 nm respectively,
d) monitoring on excitation scanning keeping the emission wavelength at 330 nm, the observed excitation maxima are at 228.2 nm and 293.8 nm and when the emission is kept at 651 nm and excitation scanning is monitored, the observed excitation maxima are at 229.2 nm and 294.8 nm respectively,
e) highly soluble in methanol, ethanol, acetone, n-butanol, fairly soluble in water, sparingly soluble in methylene chloride, di-ethyl ether, chloroform and insoluble in benzene and petroleum ether,
f) compound looses its oxidizing potency in acidic pH ranging between 4 to 5 and on keeping the solution at alkaline pH ranging between 9 to 10, the compound gradually turns brown, at pH 10 and above there is instantaneous darkening with loss of both activity and aromaticity as evidenced by UV spectroscopy,
g) the half-life of the oxidant, when stored in the solid state at a temperature ranging between 25xc2x0 C. to 30xc2x0 C. under darkness is about 48 hours as determined by its oxidative potency, but in solution of 50 mM potassium phosphate buffer of pH 7.4 at 25xc2x0 C. to 30xc2x0 C. the half life is about 90 minutes,
h) reduces ferricytochrome c and ferric chloride,
i) oxidizes ascorbic acid, proteins and DNA, and
j) the melting point is 162xc2x0 C.,
Still another embodiment of the invention, wherein p-benzosemiquinone present in cs solution is quantitatively assayed by HPLC with a UV detector using a 25 cm reverse phase ODS column and using a mixture of water and methanol (95:5 v/v) as a mobile phase, at a wave length of 288 nm, flow rate of 0.8 ml/min, at a temperature of about 25xc2x0 C. and at a pressure of about 147 Kgf/cm2 and having a retention time of 13.46 min.
Yet another embodiment of the invention, wherein the said p-benzosemiquinone isolated from the whole cs solution is responsible for the major cause of oxidative damage of proteins.
Yet another embodiment of the invention, wherein p-benzosemiquinone, the cs oxidant is responsible for the oxidative damage of DNA.
Still yet another embodiment of the invention, wherein the damage of proteins caused by p-benzosemiquinone present in cs solution is quantitatively determined by measuring protein carbonyl formation by reacting the protein with p-benzosemiquinone obtained from the cs solution, followed by reaction with 2,4 dinitrophenyl hydrazine (DNPH) and finally measuring the absorbance at a wave length of 390 nm.
In yet another embodiment of the invention, wherein the damage of proteins caused by p-benzosemiquinone present in cs solution is quantitatively determined by measuring oxidative degradation of guinea pig tissue microsomal proteins by reacting the said protein with p-benzosemiquinone present in cs solution followed by SDS-PAGE and densitometric scanning.
Yet another embodiment of invention, wherein the protein used for the assay of oxidative damages of protein is selected from the group consisting of BSA and guinea pig lung microsomal proteins
Yet another embodiment of the invention, wherein the BSA oxidation produced by the whole cs solution is effected by the p-benzosemiquinone present in the cs solution.
In yet another embodiment of the invention, the BSA oxidation produced by the cs oxidant as evidenced by nmoles of carbonyl formed per mg BSA is 9.56xc2x10.14 in comparison to 7.53xc2x10.34 produced by the whole cs solution.
In yet another embodiment of the invention, the BSA oxidation produced by the cs oxidant is evidenced by nmoles of carbonyl formed per mg BSA is 9.56xc2x10.14 in comparison to 8.16xc2x10.24 produces by the aqueous extract of cigarette smoke.
In yet another embodiment of the invention, the BSA oxidation produced by the cs oxidant is evidenced by nmoles of carbonyl formed per mg BSA is 9.56xc2x10.14 in comparison to 9.23xc2x10.14 produces by the TLC purified aqueous extract of cigarette smoke.
In yet another embodiment of the invention, the oxidative degradation of guinea pig tissue microsomal proteins produced by the p-benzosemiquinone solution is evidenced by SDS-PAGE is comparable to that produced by the whole cs solution.
In yet another embodiment of the invention, wherein the said method is used for quantitative determination of cs oxidant p-benzosemiquinone in cigarettes based on the tar content of the particular commercial brand of the cigarette.
In yet another embodiment of the invention, wherein the said method is used for quantitative determination of cs oxidant p-benzosemiquinone in cigarettes based on toxicity level of the particular commercial brand of the cigarette.
One more embodiment of the invention relates to a method for the prevention of cigarette smoke induced protein oxidation in vitro, said method comprises inhibiting the BSA oxidation by using a chemical compound or agent selected from the group consisting of ascorbic acid, sodium dithionite, tartaric acid, citric acid, oxalic acid, succinic acid, histidine, lysine, thiourea, glutathione, black tea extract, green tea extract, catechin, epigallocatechin and epicatechin.
In another embodiment of the invention, wherein ascorbic acid inhibits BSA oxidation up to 76% at a concentration of about 100 xcexcM.
In still another embodiment of the invention, wherein Sodium dithionite inhibits BSA oxidation up to 97% at a concentration of about 2 mM.
Still another embodiment of the invention, wherein tartaric acid inhibits BSA oxidation up to 75% at a concentration ranging between 500 xcexcM and 1 mM.
In yet another embodiment of the invention, wherein citric acid inhibits BSA oxidation up to 75% at a concentration ranging between 500 xcexcM and 1 mM.
In yet another embodiment of the invention, wherein oxalic acid inhibits BSA oxidation up to 53% at a concentration of about 500 xcexcM.
In yet another embodiment of the invention, wherein succinic acid inhibits BSA oxidation up to 60% at a concentration of about 1 mM.
In yet another embodiment of the invention, wherein histidine acid inhibits BSA oxidation up to 67% at a concentration of about 1 mM.
In another embodiment of the invention, wherein black tea extract inhibits BSA oxidation up to 50% at a concentration of about 2.5 mg.
Yet another embodiment of the invention, wherein catechin inhibits BSA oxidation up to 54% at a concentration range of about 750 xcexcg.
Yet another embodiment of the invention, wherein epigallocatechin inhibits BSA oxidation up to 95% at a concentration of about 140 xcexcg.
Yet another embodiment of the invention, wherein epicatechin inhibits BSA oxidation up to 50% at a concentration of about 50 xcexcg.
Yet another embodiment of the invention, wherein green tea extract inhibits BSA oxidation up to 50% at a concentration of about 2.5 mg.
Yet another embodiment of the invention, wherein lysine inhibits BSA oxidation up to 35% at a concentration of about 1 mM.
Yet another embodiment of the invention, wherein thiourea inhibits BSA oxidation up to 52% at a concentration of about 10 mM.
Yet another embodiment of the invention, wherein glutanthione inhibits BSA oxidation up to 37% at a concentration of about 1 mM.
One more embodiment of the invention relates antidotes for the harmful effect caused by the cigarette smoke oxidant which are selected from the group consisting of ascorbic acid, sodium dithionite, tartaric acid, citric acid, oxalic acid, succinic acid, histidine, lysine, thiourea, glutathione, black tea extract, green tea extract, catechine, epigallocatechin and epicatechin.
Still another embodiment of the invention relates to use of the compound p-benzosemiquinone for studying the mechanism of oxidative damage-induced degenerative diseases and cancer caused by cigarette smoke producing oxidative damage to isolated protein, DNA, cultured cells or to an experimental model under laboratory conditions.
One more embodiment of the present invention relates to a method for quantitative estimation of an harmful oxidant, p-benzosemiquinone, the said method is helpful in formulating the quantity and nature of smoking material to be used in cigarette, cigar, cigarette pipes and any other convention smoking devices.
In still another embodiment of the present invention provides a method for the prevention of cigarette smoke induced protein oxidation in vitro, the said method comprises inhibiting the BSA oxidation by using a chemical compound or agent selected from the group consisting of ascorbic acid, sodium dithionite, tartaric acid, citric acid, oxalic acid, succinic acid, histidine, lysine, thiouria, glutathione, black tea extract, green tea extract, catechine, epigallocatechin and epicatechin, the said inhibition of BSA oxidation is shown below in a tabular form.
Protection of cs-oxidant-induced albumin oxidation by different chemical agents
In still another embodiment the compound p-benzosemiquinone is useful in effecting oxidative damage to isolated protein, DNA or cultured cells under laboratory conditions to enable study of the mechanism of oxidative damage-induced degenerative diseases and cancer caused by cigarette smoke.
The present invention is described with reference to examples herein below, which are illustrative only and should not be construed to limit the scope of present invention in any manner.